This invention is directed to stained lymphocytes and a method of preparing same by using a single, substantially pure oxazine dye for the cytological preparation of a biopsy specimen of hematopoietic origin. More specifically, this invention relates to the use of a single oxazine dye for differentiating, identifying and enumerating lymphocytes among a plurality of cells of hematopoietic origin selected from the group consisting of blood cells, bone marrow cells and lymph node cells. Peripheral blood lymphocytes displayed a plurality of sizes and colors when exposed first to a fixative solution of an oxazine dye (C.I. basic blue 141), then to an aqueous alkaline solution of the same dye and subsequently rinsed in a neutral HEPES buffer containing small but effective amounts of various metal compounds. Purified lymphocyte subpopulations obtained with a cell sorter, showed that T helper cells (CD4) were small and their nuclei and cytoplasm stained deep blue. T cytotoxic/suppressor cells (CDS) were larger than T helper cells, their nuclei stained pale green or blue green and their cytoplasm contained a cluster of magenta colored granules. Used in the manner described, basic blue 141 is a means of identifying and differentiating CD4 and CD8 cells under the ordinary light microscope without using monoclonal antibodies or fluorescence.
In the prior art, there are various known methods for counting and classifying leukocytes. For example, the cytotechnician microscopically views a biopsy specimen prepared on an ordinary microscope slide that has been stained with Romanowsky stains. The cytotechnician examines the stained leukocytes and classifies them according to cell type. This method, however, is time consuming and has the disadvantage of not being reliable with respect to counting and classifying the less abundant cells. In a typical automated system, a biopsy specimen is stained in the usual manner with a Romanowsky stain and then scanned mechanically under a microscope provided with an electronic image tube. When the leukocyte comes in view of the image tube, the slide is stopped. An image analyzing computer connected to the image tube classifies the leukocyte according to its cell profile and cytoplasm color. Again the system has the disadvantage of being time consuming and costly. The use of a single specific oxazine dye (BB141) is an improvement over the Romanowsky stains even though mixtures of dyes are still the basis for morphological hematology and are being used for the identification of cells. The mixture of dyes produces the Romanowsky effect defined as a condition wherein the coloration of the cell components results from the combined action of the mixture which cannot be produced by either dye acting alone. The state of the art fails to teach the use of a single, substantially pure, commercially available stain capable of panoptically and metachromatically staining lymphocyte subpopulations in a biopsy specimen.
The identification of lymphocytes and their various subpopulations has become more important and interesting to biomedical researchers especially for diagnostic purposes. More particularly, the subpopulations of lymphocytes have been better defined and their functions in the immune system have become more clearly delineated. The important subpopulations include the B-cells, T-helper cells, T-suppressor cells, and Natural Killer (NK) cells. The B-cells are believed to be bursa derived and are involved in the synthesis of circulating immunoglobulins. The T-helper and T-suppressor cells are involved in the modulation of the immune response and in the regulation of erythropoiesis. More important, the Natural Killer cells represent the body's first line of defense against malignancy, since these cells are cytotoxic to foreign cells, and do not require the mediation of complement to effect their lysis.
In recent years the function and complexity of the T-helper and T-suppressor cell populations have received an increasing amount of attention. It is believed that the HTLV-I virus is the specific etiologic agent for adult T-cell leukemia. This virus specifically attacks the T-helper cell by entering the genetic material and "immortalizes" the cell, transforming it into a large leukemic lymphoblast with striking convolutions and indentations of the nucleus and contain a large nucleolus. The HTLV-I virus particles replicate within the T-helper cell and enter into the circulation system to infect other T-helper cells.
In contrast to the "immortalization" of the T-helper cell in the HTLV-I virus infestation, the HTLV-IV virus infects the T-helper cells specifically and in so doing destroys the cells. The HTLV-IV virus is known to be the specific causative agent of the acquired immune deficiency syndrome (AIDS). In this disorder (AIDS), it was found that the number of T-helper cells is greatly diminished because of the lethal effects of the virus, leading to a reversal of the ratio between the T-helper cell and the T-suppressor cell. Moreover, information about the antibodies to the HTLV-IV virus, and the ratio of the T-helper to the T-suppressor cells is a valuable diagnostic test for determining the presence of HTLV IV infection, and also for confirming the diagnosis of AIDS.
In the prior art, Kass teaches that the identification of T-cells and B-cells and other lymphocyte subpopulations can be accomplished by using a single organic dyestuff; see U.S. Pat. Nos. 4,400,370; 4,500,509; 4,581,223; 4,615,878; 4,714,606; 4,810,487; 4,853,210; and 5,106,744. The Kass patents disclose that Basic Orange 21 is a supravital stain and therefore must be used on living blood cells in a liquid suspension viewed in the presence of absorbance or fluorescent light. Kass also teaches that Basic Blue 41 dye, however, must be used on a fixed biopsy specimen. Here the lymphocyte subpopulations were identified on the basis of the difference in the intensity of staining and the caliber of the nuclear chromatin strands in the nucleus, as well as the presence of metachromatically staining granules in the cytoplasm.
More specifically, the differences in size and coloration of lymphocytes using mixtures of dyes have been known for nearly a century. In 1910 and 1911, Pappenheim described and illustrated lymphocytes in great detail; see Pappenheim, A., Ueber verschiedenen Typen yon Lymphozyten und Monozyten, zumm Teil im scheinbar normalen Blut; Folia Haematologica 12:26-38, 1911 and Pappenheim, A., Ferrata, A. Ueber die verschiedenen lymphoden Zellformen des normalen und pathologischen Blutes, Folia Haematol 10:78-92, 1910. Ehrlich and Lazarus described several types of lymphocytes that displayed differential coloration with the triacid staining mixture composed of methyl green, acid orange G and acid fuchsin; see Ehrlich, P. and Lazarus, A. Anemia, Histology of the blood, normal and pathologic; In Diseases of the Blood, A. Stengel, Ed. W. B. Saunders, Philadelphia. pp. 17-150, 1905. Illustrations from their chapter in Nothnagel's 1905 textbook display small lymphocytes that show dark blue staining of nucleus and cytoplasm, and other lymphocytes that are larger and stain pale green. Although Ehrlich did not speculate as to the functional significance of these different types of lymphocytes, he astutely observed that lymphocytes were not all the same. Some were large, some were small, some stained darkly and others stained pale.
More recently, identification of subpopulations of lymphocytes in specimens of blood, bone marrow, and lymph nodes has become a standardized and routine procedure in many hematology and immunopathology labs. Using monoclonal antibodies, these populations were originally characterized as T helper and T suppressor cells, reflecting their functions in the immune response. Increasingly, additional subpopulations have been identified and characterized. Based upon their surface marker characteristics, some of these subpopulations include lymphocytes having both HLADR and CD8 markers, NK or natural killer cells, and cytotoxic suppressor cells. As the repertoire of monoclonal antibodies grows, the number of subpopulations also increases, and both elucidates and makes more complex our knowledge of the immune system. Despite their biological specificity, monoclonal antibodies for lymphocyte identification are costly. When flow cytometry is used, identification of lymphocyte subpopulations remains a labor intensive and time consuming test. Furthermore, conventional cytochemical stains, such as the reactions for nonspecific esterase and acid phosphatase activities, fail to demonstrate consistent differences in staining patterns between T helper and T suppressor cells; see Armitage, R. J., Linch, D. C., Worman, C. P. and Cawley, J. C.; The morphology and cytochemistry of human T-cell subpopulations defined by monoclonal antibodies and Fc receptors, British Journal of Haematology 51:605-613, 1982 and DeWaele, M., DeMey, J., Moeremans, M., Smet, L., Broodtaerts, L, and VanCamp, B. and Cytochemical profile of immunoregulatory T-lymphocyte subsets defined by monoclonal antibodies Journal of Histochemistry and Cytochemistry 31:471-478, 1983. For these reasons investigation continues to be devoted to finding means by which these functionally defined populations can be distinguished and counted, with the expectation that measuring changes in these populations can lead to means of diagnosing, prognosticating, and following immune-mediated disorders.
Along these lines, an asymmetrical polymethine textile dye was developed into a stain for lymphocyte subpopulations using darkfield illumination; see Kass, L. Identification of lymphocyte subpopulations with a polymethine dye in the Journal of Histochemistry and Cytochemistry 36:711-715, 1988 and U.S. Pat. No. 4,810,487. Using this stain on a slide containing blood cells fixed in FAA (formalin, acetic acid, and 95% ethyl alcohol) and viewed under darkfield, T helper cells stained bright red, T suppressor cells were yellow-orange, B cells were yellow and contained yellow annular structures, and NK cells appeared green and contained prominent green granules.
Kass also disclosed that an oxazine textile dye (C.I. basic blue 141) can be used as a selective stain for neutrophils. Using an alkaline buffered solution of this stain applied to blood cells fixed in FAA fixative, the neutrophil granules stained bright crimson. The lymphocytes displayed pale green to blue green nuclei, but the cytoplasm was unstained. However, lymphocytes of different colors were not recognized; see Kass, Identification of neutrophils with an oxazine dye in the American Journal of Clinical Pathology 88:436-441, 1987. More recently, C.I. basic blue 141 was used in a buffered alkaline aqueous solution as the second reagent in a two-step stain for monocytes; see Kass, A two-step stain for monocytes using two different dyes applied in sequence (Biotechnic and Histochemistry 67:68-72, 1992).
It is evident from the state of the art that the identification and enumeration of the subpopulations of lymphocytes including the T-cells and B-cells, which are the major lymphocyte populations in man, are basic to the understanding of the immune system and the various diseases such as lymphoma, leukemias, and immunodeficiency problems. For example, B-cells represent about 10-20% of the peripheral blood lymphocytes. T-cells which are indicators of the cellular immune responses, i.e., delayed hypersensitivity and organ transplant rejection, constitute about 70-80% of the total. The lymphocyte subpopulations known as T-helper cells and T-suppressor cells, serve to regulate the antibody production and are effectors of T-cell functions. The enumeration of T-lymphocytes has diagnostic significance in certain chronic disorders where reduced numbers are found. Thus, monitoring the number of T-suppressor and T-helper cells has advanced the understanding and treatment of diseases affecting the immune system.
In accordance with this invention, crimson colored granules were made visible by a unique two-stage reaction using the stain in both the fixative and in the aqueous phases. It was found that staining occurred in T cytotoxic/suppressor cells and formed the basis for their identification and distinction from other forms of lymphocytes. The methods set forth in the above-mentioned Kass publications for obtaining coloration of the cells, required that the cells be first fixed in absolute alcohol with no stain and then stained with an aqueous buffer solution and rinsed in an aqueous solution contain metal cations with no buffer. In comparison, the colored photomicrograph of FIG. 1 shows that when peripheral blood was stained in accordance with the method of this invention, that the small dark-blue cells (nucleus and cytoplasm are both dark-blue) were identified as T-helper cells (CD4 cells). The cells with green nuclei and small maroon granules in the cytoplasm are T-cytoxic (suppressor CD8) cells and the cells with irregular blue nuclei and many red granules are neutrophils. Therefore, with a colored photomicrograph of FIG. 1, one would be able to clearly identify and therefore enumerate the lymphocytes among a plurality of other cells of hematopoietic origin.